1. Field of the Invention
The present invention relates generally to single-chain antigen-binding molecules capable of glycosylation. More specifically, the invention relates to antigen-binding proteins having Asn-linked glycosylation sites capable of attaching a carbohydrate moiety. The invention also relates to multivalent antigen-binding molecules capable of glycosylation. The invention further relates to glycosylated antigen-binding molecules capable of polyalkylene oxide conjugation. Compositions of, genetic constructions for, methods of use, and methods for producing glycosylated antigen-binding proteins capable of polyalkylene oxide conjugation are disclosed. The invention also relates to methods for producing a polypeptide having increased glycosylation and the polypeptide produced by the described methods.
2. Description of the Background Art
Antibodies are proteins generated by the immune system to provide a specific molecule capable of complexing with an invading molecule, termed an antigen. Natural antibodies have two identical antigen-binding sites, both of which are specific to a particular antigen. The antibody molecule xe2x80x9crecognizesxe2x80x9d the antigen by complexing its antigen-binding sites with areas of the antigen termed epitopes. The epitopes fit into the conformational architecture of the antigen-binding sites of the antibody, enabling the antibody to bind to the antigen.
The IgG antibody, e.g., is composed of two identical heavy and two identical light polypeptide chains, held together by interchain disulfide bonds. The remainder of this discussion on antibodies will refer only to one pair of light/heavy chains, as each light/heavy pair is identical. Each individual light and heavy chain folds into regions of approximately 110 amino acids, assuming a conserved three-dimensional conformation. The light chain comprises one variable region (VL) and one constant region (CL), while the heavy chain comprises one variable region (VH) and three constant regions (CH1, CH2 and CH3). Pairs of regions associate to form discrete structures. In particular, the light and heavy chain variable regions associate to form an xe2x80x9cFvxe2x80x9d area which contains the antigen-binding site.
Recent advances in immunobiology, recombinant DNA technology, and computer science have allowed the creation of single polypeptide chain molecules that bind antigen. These single-chain antigen-binding molecules (xe2x80x9cSCAxe2x80x9d) or single-chain variable fragments of antibodies (xe2x80x9csFvxe2x80x9d) incorporate a linker polypeptide to bridge the individual variable regions, VL and VH, into a single polypeptide chain. A description of the theory and production of single-chain antigen-binding proteins is found in Ladner et al., U.S. Pat. Nos. 4,946,778, 5,260,203, 5,455,030 and 5,518,889. The single-chain antigen-binding proteins produced under the process recited in the above U.S. patents have binding specificity and affinity substantially similar to that of the corresponding Fab fragment A computer-assisted method for linker design is described more particularly in Ladner et al., U.S. Pat. Nos. 4,704,692 and 4,881,175, and WO 94/12520.
The in vivo properties of SCA polypeptides are different from MAbs and antibody fragments. Due to their small size, SCA polypeptides clear more rapidly from the blood and penetrate more rapidly into tissues (Milenic, D. E. et al., Cancer Research 51:6363-6371 (1991); Colcher et al., J. Natl. Cancer Inst. 82:1191 (1990); Yokota et al., Cancer Research 52:3402 (1992)). Due to lack of constant regions, SCA polypeptides are not retained in tissues such as the liver and kidneys. Due to the rapid clearance and lack of constant regions, SCA polypeptides will have low immunogenicity. Thus, SCA polypeptides have applications in cancer diagnosis and therapy, where rapid tissue penetration and clearance, and ease of microbial production are advantageous.
A multivalent antigen-binding protein has more than one antigen-binding site. A multivalent antigen-binding protein comprises two or more single-chain protein molecules. Enhanced binding activity, di- and multi-specific binding, and other novel uses of multivalent antigen-binding proteins have been demonstrated. See, Whitlow, M., et al., Protein Engng. 7:1017-1026 (1994); Hoogenboom, H. R, Nature Biotech. 15:125-126 (1997); and WO 93/11161.
Carbohydrate modifications of proteins fall into three general categories: N-linked (or Asn-linked) modification of asparagine, O-linked modification of serine or threonine and glycosyl-phosphatidylinositol derivation of the C-terminus carboxyl group. Each of these transformations is catalyzed by one or more enzymes which demonstrate different peptide sequence requirements and reaction specificities. N-linked glycosylation is catalyzed by a single enzyme, oligosaccharyl transferase (OT), and involves the co-translational transfer of a lipid-linked tetradecasaccharide (GlcNAc2-Man9-Glc3) to an asparagine side chain within a nascent polypeptide (see, Imperiali, B. and Hendrickson, T. L., Bioorganic and Med. Chem. 3:1565-1578 (1995)). The asparagine residue must reside within the tripeptide N-linked glycosylation consensus sequence Asn-Xaa-Thr/Ser (NXT/S), where Xaa can be any of the 20 natural amino acids except proline.
A natural N-linked glycosylation sequence (Asn-Val-Thr) at amino acid positions 18-20 (Kabat""s numbering) was identified in the framework-1 (FR-1) region of the light chain variable domain of a murine anti-B cell lymphoma antibody, LL-2 (Leung, S.-o. et al., J. Immunol. 154:5919-5926 (1995)). By a single Arg to Asn mutation, an N-linked glycosylation sequence similar to that of LL-2 was introduced in the FR-1 segment of a nonglycosylated, humanized anti-carcinoembryonic Ag (CEA) Ab, MN-14 (Leung, S.-O. et al., J. Immunol. 154:5919-5926 (1995), which disclosure is incorporated herein by reference).
An sFv having a C-terminus that has cross-linking means by disulfide bonds at cysteine residues has been reported (Huston et al., U.S. Pat. No. 5,534,254). A monoclonal antibody has also been reported that is covalently bound to a diagnostic or therapeutic agent through a carbohydrate moiety at an Asn-linked glycosylation site at about amino acid position 18 of the VL region (Hansen et al., U.S. Pat. No. 5,443,953). Binding studies of an anti-dextran antibody that is Asn-linked glycosylated in the VH chain have been performed which show that slight changes in the position of the Asn-linked carbohydrate moiety in the VH region result in substantially different effects on antigen binding (Wright et al., EMBO J. 10:2717-2723 (1991)). It has also been shown that glycosylation at position 19 within the VH region of an sFv enhanced expression of the overall amount of sFv intracellularly, of which approximately half was glycosylated (Greenman, J., et al., J. Immunol. Methods 194:169-180 (1996)), and enhanced synthesis and secretion of the glycosylated sFv over the nonglycosylated sFv (Jost, C. R., et al., J. Biol. Chem. 269:26267-26273 (1994)). Co et al., U.S. Pat. No. 5,714,350, relates to increasing binding affinity of an antibody by eliminating a glycosylation site.
The covalent attachment of strands of a polyalkylene glycol or polyalkylene oxide to a polypeptide molecule is disclosed in U.S. Pat. No. 4,179,337 to Davis et al., as well as in Abuchowski and Davis xe2x80x9cEnzymes as Drugs,xe2x80x9d Holcenberg and Roberts, Eds., pp. 367-383, John Wiley and Sons, New York(1981), and Zalipsky et al., WO 92/16555. These references disclosed that proteins and enzymes modified with polyethylene glycols have reduced immunogenicity and antigenicity and have longer lifetimes in the bloodstream, compared to the parent compounds. The resultant beneficial properties of the chemically modified conjugates are very useful in a variety of therapeutic applications.
To effect covalent attachment of polyethylene glycol (PEG) and similar poly(alkylene oxides) to a molecule, the hydroxyl end groups of the polymer must first be converted into reactive functional groups. This process is frequently referred to as xe2x80x9cactivationxe2x80x9d and the product is called xe2x80x9cactivated PEG.xe2x80x9d
Hydrazides readily form relatively stable hydrazone linkages by condensation with aldehydes and ketones (Andresz, H. et al., Makromol. Chem. 179:301 (1978)). This property has been used extensively for modification of glycoproteins through oxidized oligosaccharide moieties (Wilchek, M. and Bayer, E. A., Meth. Enzymol. 138:429 (1987)).
Activated PEG-hydrazide allows it to react with an aldehyde group. Aldehyde is normally absent on the polypeptide chain of a protein. However, if a protein contains carbohydrate moieties, then the carbohydrate can be activated to provide a reactive aldehyde group by oxidation of the sugar ring such as mannose. Methods for activation of immunoconjugates are described in Sela et al., Immunoconjugates, Vogel ed., Oxford University Press (1987). In this way, PEG-hydrazide can be conjugated covalently to the protein via the carbohydrate structure. Zalipsky, S., et al., WO 92/16555, describes PAO covalently bonded to an oxidized carbohydrate moiety of the glycopolypeptide by a linkage containing a hydrazide or hydrazone functional group bound to the polymer. The oxidation of the carbohydrate moiety produces reactive aldehydes. The hydrazone linkage is formed by reacting an acyl hydrazine derivative of the polymer containing the peptide sequence with these aldehyde groups.
The prior art has activated the hydroxyl group of PEG with cyanuric chloride and the resulting compound is then coupled with proteins (Abuchowski et al., J. Biol. Chem. 252:3578 (1977); Abuchowski and Davis, supra (1981)). However, there are disadvantages in using this method, such as the toxicity of cyanuric chloride and its non-specific reactivity for proteins having functional groups other than amines, such as free essential cysteine or tyrosine residues.
In order to overcome these and other disadvantages, alternative activated PEGs, such as succinimidyl succinate derivatives of PEG (xe2x80x9cSS-PEGxe2x80x9d), have been introduced (Abuchowski et al., Cancer Biochem. Biophys. 7:175-186 (1984)). It reacts quickly with proteins (30 minutes) under mild conditions yielding active yet extensively modified conjugates.
Zalipsky, in U.S. Pat. No. 5,122,614, disclosed poly(ethylene glycol)-N-succinimide carbonate and its preparation. This form of the polymer was said to react readily with the amino groups of proteins, as well as low molecular weight peptides and other materials that contain free amino groups.
Other linkages between the amino groups of the protein, and the PEG are also known in the art, such as urethane linkages (Veronese et al., Appl. Biochem. Biotechnol. 11:141-152 (1985)), carbamate linkages (Beauchamp et al., Analyt. Biochem. 131:25-33 (1983)), and others.
Polyalkylene oxide modification of sFvs is disclosed in U.S. Provisional Patent Application No. 60/050,472, filed Jun. 23, 1997, which disclosure is incorporated herein by reference.
The activated polymers can also be reacted with a therapeutic agent having nucleophilic functional groups that serve as attachment sites. One nucleophilic functional group commonly used as an attachment site is the xcex5-amino groups of lysines. Free carboxylic acid groups, suitably activated carbonyl groups, oxidized carbohydrate moieties and mercapto groups have also been used as attachment sites.
Conjugation of poly(ethylene glycol) or poly(alkylene oxide) with small organic molecules is described in Greenwald, R. B., Exp. Opin. Ther. Patents 7:601-609 (1997), Enzon Inc., WO 95/11020, and Enzon Inc., WO 96/23794, which disclosures are all incorporated herein by reference. Compositions based on the use of various linker groups between the PEG ballast and the active drug are described in WO 96/23794.
The invention is directed to a single-chain antigen-binding polypeptide capable of glycosylation, comprising:
(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and
(c) a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site,
wherein the single-chain antigen-binding polypeptide has at least one tripeptide Asn-linked glycosylation sequence comprising Asn-Xaa-Yaa, wherein Xaa is an amino acid other than proline and Yaa is threonine or serine, wherein the tripeptide glycosylation sequence is capable of attaching a carbohydrate moiety at the Asn residue located at a position selected from the group consisting of (i) the amino acid position 11, 12, 13, 14 or 15 of the light chain variable region; (ii) the amino acid position 77, 78 or 79 of the light chain variable region; (iii) the amino acid position 11, 12, 13, 14 or 15 of the heavy chain variable region; (iv) the amino acid position 82B, 82C or 83 of the heavy chain variable region; (v) any amino acid position of the peptide linker; (vi) adjacent to the C-terminus of the second polypeptide (b); and (vii) combinations thereof, wherein the glycosylated single-chain antigen-binding polypeptide is capable of binding an antigen.
The invention is further directed to a polynucleotide encoding a single-chain antigen-binding polypeptide capable of glycosylation, comprising:
(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and
(c) a peptide linker linking the first and second polypeptides (a) and
(b) into a single chain polypeptide having an antigen binding site,
wherein the single-chain antigen-binding polypeptide has at least one tripeptide Asn-linked glycosylation sequence comprising Asn-Xaa-Yaa, wherein Xaa is an amino acid other than proline and Yaa is threonine or serine, wherein the tripeptide glycosylation sequence is capable of attaching a carbohydrate moiety at the Asn residue located at a position selected from the group consisting of (i) the amino acid position 11, 12, 13, 14 or 15 of the light chain variable region; (ii) the amino acid position 77, 78 or 79 of the light chain variable region; (iii) the amino acid position 11, 12, 13, 14 or 15 of the heavy chain variable region; (iv) the amino acid position 82B, 82C or 83 of the heavy chain variable region; (v) any amino acid position of the peptide linker; (vi) adjacent to the C-terminus of the second polypeptide (b); and (vii) combinations thereof, wherein the glycosylated single-chain antigen-binding polypeptide is capable of binding an antigen.
The polynucleotide may be DNA or RNA.
The invention is directed to a replicable cloning or expression vehicle comprising the above described DNA sequence. The invention is also directed to such vehicle which is a plasmid. The invention is further directed to a host cell transformed with the above described DNA. The host cell may be a bacterial cell, a yeast cell or other fungal cell, an insect cell or a mammalian cell line. A preferred host is Pichia pastoris. 
The invention is directed to a method of producing a single-chain antigen-binding polypeptide capable of glycosylation, comprising:
(a) providing a first polynucleotide encoding a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
(b) providing a second polynucleotide encoding a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and
(c) linking the first and second polynucleotides (a) and (b) with a third polynucleotide encoding a peptide linker into a fourth polynucleotide encoding a single chain polypeptide having an antigen binding site,
wherein the single-chain antigen-binding polypeptide has at least one tripeptide Asn-linked glycosylation sequence comprising Asn-Xaa-Yaa, wherein Xaa is an amino acid other than proline and Yaa is threonine or serine, wherein the tripeptide glycosylation sequence is capable of attaching a carbohydrate moiety at the Asn residue located at a position selected from the group consisting of (i) the amino acid position 11, 12, 13, 14 or 15 of the light chain variable region; (ii) the amino acid position 77, 78 or 79 of the light chain variable region; (iii) the amino acid position 11, 12, 13, 14 or 15 of the heavy chain variable region; (iv) the amino acid position 82B, 82C or 83 of the heavy chain variable region; (v) any amino acid position of the peptide linker; (vi) adjacent to the C-terminus of the second polypeptide (b); and (vii) combinations thereof, wherein the glycosylated single-chain antigen-binding polypeptide is capable of binding an antigen; and
(d) expressing the single-chain antigen-binding polypeptide of (c) in the host, thereby producing a single-chain antigen-binding polypeptide capable of glycosylation.
In the method as according to the invention, the host cell is capable of catalyzing glycosylation. The host cell is a plant cell, a bacterial cell, a yeast cell or other fungal cell, an insect cell or a mammalian cell line. A preferred host cell is Pichia pastoris. 
The invention is further directed to a multivalent single-chain antigen-binding protein, comprising two or more single-chain antigen-binding polypeptides, each single-chain antigen-binding polypeptide comprising:
(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and
(c) a peptide linker linking the first and second polypeptides (a) and
(b) into a single chain polypeptide having an antigen binding site, wherein the single-chain antigen-binding polypeptide has at least one tripeptide Asn-linked glycosylation sequence comprising Asn-Xaa-Yaa, wherein Xaa is an amino acid other than proline and Yaa is threonine or serine, wherein the tripeptide glycosylation sequence is capable of attaching a carbohydrate moiety at the Asn residue located at a position selected from the group consisting of (i) the amino acid position 11, 12, 13, 14 or 15 of the light chain variable region; (ii) the amino acid position 77, 78 or 79 of the light chain variable region; (iii) the amino acid position 11, 12, 13, 14 or 15 of the heavy chain variable region; (iv) the amino acid position 82B, 82C or 83 of the heavy chain variable region; (v) any amino acid position of the peptide linker; (vi) adjacent to the C-terminus of the second polypeptide (b); and (vii) combinations thereof, wherein the glycosylated single-chain antigen-binding polypeptide is capable of binding an antigen.
In the above described embodiments of the invention, the tripeptide glycosylation sequence may be capable of attaching a carbohydrate moiety at the Asn residue located at a position selected from the group consisting of (ixe2x80x2) the amino acid position 12 of the light chain variable region; (iixe2x80x2) the amino acid position 77 of the light chain variable region; (iiixe2x80x2) the amino acid position 13 of the heavy chain variable region; (ivxe2x80x2) the amino acid position 82B of the heavy chain variable region; (vxe2x80x2) the amino acid position 2 of the peptide linker; (vixe2x80x2) adjacent to the C-terminus of the second polypeptide (b); and (viixe2x80x2) combinations thereof, wherein the glycosylated single-chain antigen-binding polypeptide is capable of binding an antigen.
In the above described embodiments of the invention, at least one single-chain antigen-binding polypeptide may have at least two tripeptide glycosylation sequences in tandem such that the Asn residues are separated by two amino acid residues and/or at least one set of two overlapping tripeptide glycosylation sequences such that the Asn residues are adjacent. At least one single-chain antigen-binding polypeptide may have three tripeptide glycosylation sequences in tandem. At least one single-chain antigen-binding polypeptide may have at least two sets of two tandem tripeptide glycosylation sequences and at least two sets of two overlapping tripeptide glycosylation sequences.
Also in the above described embodiments of the invention, the Asn residue of the tripeptide glycosylation sequence may be attached to a carbohydrate moiety. The carbohydrate moiety may further be conjugated to polyalkylene oxide. The carbohydrate and/or polyalkylene moieties may be conjugated to one or plurality of peptide, lipid, nucleic acid, drug, toxin, chelator, boron addend or detectable label molecule(s). The carbohydrate and/or polyalkylene oxide moieties may be conjugated to a carrier having one or plurality of peptide, lipid, nucleic acid, drug, toxin, chelator, boron addend or detectable label molecule(s) bound to the carrier.
In the above described embodiments of the invention, the C-terminus of the second polypeptide (b) may be the native C-terminus of the second polypeptide (b). In the alternative, the C-terminus of the second polypeptide (b) may comprise a deletion of one or plurality of amino acid residue(s), such that the remaining N-terminus amino acid residues of the second polypeptide are sufficient for the glycosylated polypeptide to be capable of binding an antigen. In the alternative, the C-terminus of the second polypeptide may comprise an addition of one or plurality of amino acid residue(s), such that the glycosylated polypeptide is capable of binding an antigen. In one embodiment, the Asn residue of the glycosylation sequence may be located adjacent to any of the above mentioned C-terminus of the second polypeptide and the glycosylation sequence may be followed by at least one amino acid residue. In the alternative, the glycosylation sequence may be followed by two, three, four or five amino acid residues.
In a preferred embodiment of the invention, the first polypeptide (a) may comprise the antigen binding portion of the variable region of an antibody light chain and the second polypeptide (b) comprises the antigen binding portion of the variable region of an antibody heavy chain.
The invention is also directed to a method of detecting an antigen suspected of being in a sample, comprising:
(a) contacting the sample with the glycosylated polypeptide or multivalent protein of the invention, wherein the carbohydrate moiety is conjugated to one or plurality of detectable label molecule(s), or conjugated to a carrier having one or plurality of detectable label molecule(s) bound to the carrier, and
(b) detecting whether the glycosylated single-chain antigen-binding polypeptide has bound to the antigen.
The invention is further directed to a method of imaging the internal structure of an animal, comprising administering to the animal an effective amount of the glycosylated polypeptide or multivalent protein of the invention, wherein the carbohydrate moiety is conjugated to one or plurality of detectable label or chelator molecule(s), or conjugated to a carrier having one or plurality of detectable label or chelator molecule(s) bound to the carrier, and measuring detectable radiation associated with the animal. Animal includes human and nonhuman.
The invention is also directed to a method for treating a targeted disease, comprising administering an effective amount of a composition comprising the glycosylated polypeptide or multivalent protein of the invention and a pharmaceutically acceptable carrier vehicle, wherein the carbohydrate moiety is conjugated to one or plurality of peptide, lipid, nucleic acid, drug, toxin, boron addend or radioisotope molecule(s), or conjugated to a carrier having one or plurality of drug, toxin, boron addend or radioisotope molecule(s) bound to the carrier.
The above described methods may be facilitated with the glycosylated polypeptide or multivalent protein of the invention, which is conjugated to polyalkylene oxide which may also be conjugated to one or plurality of peptide, lipid, nucleic acid, drug, toxin, chelator, boron addend or detectable label molecule(s).
The invention also relates to (1) a method of producing a polypeptide having increased glycosylation, comprising: (a) providing to a polynucleotide encoding the polypeptide at least two tripeptide Asn-linked glycosylation sequences, wherein each tripeptide glycosylation sequence comprises Asn-Xaa-Yaa, wherein Xaa is an amino acid other than proline and Yaa is threonine or serine, and wherein the tripeptide glycosylation sequences are in tandem such that the Asn residues are separated by two amino acid residues; and (b) expressing the polynucleotide in a host cell capable of attaching a carbohydrate moiety at the Asn residues, and (2) a polypeptide having increased glycosylation produced by the described process.
The invention further relates to (1) a method of producing a polypeptide having increased glycosylation, comprising: (a) providing to a polynucleotide encoding the polypeptide at least one set of two tripeptide Asn-linked glycosylation sequences, wherein each tripeptide glycosylation sequence comprises Asn-Xaa-Yaa, wherein Xaa is an amino acid other than proline and Yaa is threonine or serine, and wherein the two tripeptide glycosylation sequences overlap such that the Asn residues are adjacent; and (b) expressing the polynucleotide in a host cell capable of attaching a carbohydrate moiety at the Asn residues, and (2) a polypeptide having increased glycosylation produced by the described process.
The invention also relates to (1) a method of producing a polypeptide having increased glycosylation, comprising: (a) providing to a polynucleotide encoding the polypeptide at least two tripeptide Asn-linked glycosylation sequences, wherein each tripeptide glycosylation sequence comprises Asn-Xaa-Yaa, wherein Xaa is an amino acid other than proline and Yaa is threonine or serine, and wherein the tripeptide glycosylation sequences are in tandem such that the Asn residues are separated by two amino acid residues; (b) providing to the polynucleotide at least one set of two tripeptide Asn-linked glycosylation sequences, wherein the two tripeptide glycosylation sequences overlap such that the Asn residues are adjacent; and (c) expressing the polynucleotide in a host cell capable of attaching a carbohydrate moiety at the Asn residues, and (2) a polypeptide having increased glycosylation produced by the described process.